In birds and mammals, all mesoderm cells are generated from the primitive streak. Nascent mesoderm cells contain unique dorsoventral (D/V) identities according to their relative ingression position along the streak. Molecular mechanisms controlling this initial phase of mesoderm diversification are not well understood. Using the chick model, we generated high-quality transcriptomic datasets of different streak regions and analyzed their molecular heterogeneity. Fifteen percent of expressed genes exhibit differential expression levels, as represented by two major groups (dorsal to ventral and ventral to dorsal). A complete set of transcription factors and many novel genes with strong and region-specific expression were uncovered. Core components of BMP, Wnt and FGF pathways showed little regional difference, whereas their positive and negative regulators exhibited both dorsal-to-ventral and ventral-to-dorsal gradients, suggesting that robust D/V positional information is generated by fine-tuned regulation of key signaling pathways at multiple levels. Overall, our study provides a comprehensive molecular resource for understanding mesoderm diversification in vivo and targeted mesoderm lineage differentiation in vitro.
Congenital scoliosis (CS) is a common vertebral malformation. Spondylocostal dysostosis (SCD) is a rare skeletal dysplasia characterised by multiple vertebral malformations and rib anomalies. In a previous study, a compound heterozygosity for a null mutation and a risk haplotype composed by three single-nucleotide polymorphisms in TBX6 have been reported as a disease-causing model of CS. Another study identified bi-allelic missense variants in a SCD patient. The purpose of our study is to identify TBX6 variants in CS and SCD and examine their pathogenicity.We recruited 200 patients with CS or SCD and investigated TBX6 variants. We evaluated the pathogenicity of the variants by in silico prediction and in vitro experiments.We identified five 16p11.2 deletions, one splice-site variant and five missense variants in 10 patients. In vitro functional assays for missense variants identified in the previous and present studies demonstrated that most of the variants caused abnormal localisation of TBX6 proteins. We confirmed mislocalisation of TBX6 proteins in presomitic mesoderm cells induced from SCD patient-derived iPS cells. In induced cells, we found decreased mRNA expressions of TBX6 and its downstream genes were involved in somite formation. All CS patients with missense variants had the risk haplotype in the opposite allele, while a SCD patient with bi-allelic missense variants did not have the haplotype.Our study suggests that bi-allelic loss of function variants of TBX6 cause a spectrum of phenotypes including CS and SCD, depending on the severity of the loss of TBX6 function.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Innate pluripotency of mouse embryos transits from naive to primed state as the inner cell mass differentiates into epiblast. In vitro, their counterparts are embryonic (ESCs) and epiblast stem cells (EpiSCs), respectively. Activation of the FGF signaling cascade results in mouse ESCs differentiating into mEpiSCs, indicative of its requirement in the shift between these states. However, only mouse ESCs correspond to the naive state; ESCs from other mammals and from chick show primed state characteristics. Thus, the significance of the naive state is unclear. In this study, we use zebra finch as a model for comparative ESC studies. The finch blastoderm has mESC-like properties, while chick blastoderm exhibits EpiSC features. In the absence of FGF signaling, finch cells retained expression of pluripotent markers, which were lost in cells from chick or aged finch epiblasts. Our data suggest that the naive state of pluripotency is evolutionarily conserved among amniotes. https://doi.org/10.7554/eLife.07178.001 eLife digest In animals, stem cells divide to give rise to other cells that have specialized roles in the body. ‘Pluripotent’ stem cells—which are able to produce cells of any type—can be obtained from young mouse embryos. Once grown in the laboratory, these cells are called naive embryonic stem cells (ESCs) and their discovery has been vitally important for understanding how mammals develop. ESCs also have considerable medical potential because they could be used to repair or replace tissues that have been lost to injury or disease. A family of proteins called fibroblast growth factors (FGFs) triggers naive ESCs to mature into another class of stem cell that are ‘primed’ to only produce particular types of cells. Curiously, the stem cells that have been collected from other mammal embryos are already in this primed state. Therefore, biologists wonder whether the naive state is exclusive to mice embryos, or whether it is present in other animals but has so far remained undetected. The development of chick and other bird embryos shares many parallels with that of mammals. However, embryos in chicken eggs do not contain naive ESCs. It is possible that this is due to chicken eggs being laid when the embryos have reached a later stage in development where the naive stem cells have already matured into the primed cells. Here, Mak et al. compared the stem cells in chick embryos to those from another bird called the zebra finch. The experiments show that the finch embryos contain stem cells that share several features with mouse ESCs. In particular, these finch cells continue to express genes that are required for the naive state to be maintained in the absence of FGF. On the other hand, these genes are switched off in cells from chick embryos and in older zebra finch stem cells. Mak et al.'s findings show that finch eggs are laid at an earlier stage of embryo development than chicken eggs. The experiments also suggest that both birds and mammals have naive pluripotent stem cells during the early stages of embryo development. In future, the zebra finch could be used as a model to study stem cells and other aspects of animal development. https://doi.org/10.7554/eLife.07178.002 Introduction The successful isolation and in vitro culture of embryonic stem cells (ESC) from mouse embryos have enabled technological breakthroughs and revolutionized our understanding of the molecular mechanisms regulating mammalian development (Evans, 2011). However, similar applications to other species have been lacking. One conceptual difficulty has been linking the innate pluripotency of the embryo with the characteristics of cultured stem cells, raising the speculation that mESCs may be solely a result of in vitro manipulations (Pauklin et al., 2011). In the mouse, ESCs are at naive state (Nichols and Smith, 2009), which recent evidence suggests is most similar to cells from embryonic day (E) 4–4.5 of mouse development (Boroviak et al., 2014). Cells taken from this stage can give rise to derivatives of all three germ layers as well as germ cells. These cells, both in their native naive state and in culture, express genes associated with pluripotency such as Oct3/4 (Pou5f1), Sox2, and Nanog (2). Using defined media that includes the presence of either an inhibitor of FGF signaling or its downstream Erk/MAP kinase transduction pathway, mouse ESCs (mESCs) can be propagated while maintaining the expression of these pluripotency markers (Ying et al., 2008). A second pluripotent cell type in the mouse, epiblast stem cells (mEpiSCs), is derived from embryos that are later in development (E5.5) and is in what has been termed, the primed state (Brons et al., 2007). These cells have a more limited potency and require different culture condition for in vitro propagation (Lanner and Rossant, 2010), with a dependency on FGF-mediated ERK activation for the maintenance of pluripotent gene expression. Pluripotent ESCs from other mammalian organisms, such as human (Thomson et al., 1998; Schatten et al., 2005), and from non-mammalian amniotes, such as chick (Pain et al., 1996), share this requirement for ERK signaling (Tesar et al., 2007). Hence, the primed state of pluripotency is evolutionarily conserved in mammalian and non-mammalian amniotes. However, the naive state has so far only been confirmed in the mouse (Ying et al., 2008) and rat (Buehr et al., 2008; Li et al., 2008; Chen et al., 2013b), raising the possibility that this state is not conserved among the amniotes. More recent reports suggested that with specific reprogramming factors and culture conditions such a naive state may also exist for human ESCs, although the exact nature of these naive-type human cells is under debate (Takashima et al., 2014; Theunissen et al., 2014; Ware et al., 2014). Identifying the naive state of embryogenesis in other species is therefore central to our conceptual understanding of pluripotent stem cells. A comparative embryology approach to address this question should include non-mammalian amniotes. These include avian species, which share key molecular and cellular features of epiblast morphogenesis with the mammals (Sheng, 2014), yet are evolutionarily distant enough to serve as an outgroup. As in all amniotes, fertilization of avian oocytes takes place internally and avian embryos undergo some development prior to egg-laying (oviposition). The most widely used avian developmental models are chicken (Gallus gallus), quail (Coturnix japonica), and zebra finch (Taeniopygia guttata). However, chicken embryos at oviposition are already at a late blastula/early gastrulation stage (Eyal-Giladi and Kochav (EGK) stage X) (Eyal-Giladi and Kochav, 1976). These embryos have a columnar epithelialized epiblast overlying scattered hypoblasts and are thus morphologically similar to E5.5 mouse embryo, later than the stage at which mESCs can be derived. The Japanese quail embryos are laid at a stage later than the chicken embryos (Sellier et al., 2006), while the ovipositional stage of zebra finch embryos is unclear. Early-staged avian embryos can generate cells that show some of the characteristics of mammalian ES cells (Jean et al., 2015) after the introduction of reprogramming factors (Rossello et al., 2013; Dai et al., 2014) or after the manipulation of culture conditions (Pain et al., 1996; Jean et al., 2013). However, it was not clear how the pluripotency generated be exogenous factors related to the pluripotent state of cells in the embryo. We decided to investigate the early development of the zebra finch (hereafter referred to as the finch) in more detail. The finches are a model system commonly used in neurobiological studies of social behavior (Brazas and Shimizu, 2002; Svec et al., 2009), vocalization, and learning (Jarvis, 2004; Petkov and Jarvis, 2012). These studies have led to an increased focus on the developmental neurobiology of zebra finch (Charvet and Striedter, 2009; Chen et al., 2013a), and in turn the embryology of the finch (Murray et al., 2013). In addition, the finch genome has been sequenced (Warren et al., 2010) and their relatively small adult body sizes (4–7 times smaller than adult chickens) and shorter generation time (2–3 months for finches vs 4–6 months for the chickens) makes it feasible to breed them within a normal laboratory setting. Thus, we asked whether finch embryos could be used for early embryogenesis and ESCs studies, complementing existing studies (Rossello et al., 2013), which together may lead to potential technological breakthroughs that facilitate genetic and functional investigations in this model organism. Here, we report the first molecular characterization of ovipositional finch blastoderms by quantitative RT-PCR, immunohistochemical staining, and in situ hybridization. Our results suggested that finch embryos are laid at stage EGK-VI to EGK-VIII, much earlier than the chicken embryos, and are morphologically more similar to the E4–E4.5 mouse embryos from which mESCs can be derived. Cells derived from the finch blastoderm at oviposition and cultured in the presence of a MEK inhibitor and Leukemia inhibitory factor (LIF) retained expression of the pluripotent markers; Nanog, PouV expression, and alkaline phosphatase (AP) activity. In contrast, chicken cells taken from newly laid embryos and cultured under the same conditions did not produce Nanog, PouV, or AP-positive aggregates. Our data suggest that birds and mammals share a common regulatory mechanism in the maintenance of pluripotency. Finch embryos are ideally suited for the establishment and characterization of avian ESCs, and the incorporation of recent technical improvements (Dai et al., 2014) could lead to the finch becoming a tractable avian model for genetics and regenerative medicine. Results Finch oviposition is at EGK-VI prior to subgerminal cavity expansion Avian embryos undergo varying degrees of intrauterine development prior to oviposition (egg-laying). Chick oviposition is at the late blastula/early gastrula stage (EGK-X to EGK-XI). In other Galloanserae, oviposition ranges from EGK-VII (Turkey [Gupta and Bakst, 1993] and Duck [Sellier et al., 2006]) to EGK-XI (the quail [Stepinska and Olszanska, 1983]). Ratite embryos are laid at EGK-X, similar to the chick embryos (Nagai et al., 2011). The oviposition stage of Passerine (songbird) species has not been carefully investigated, although gross morphology of newly laid embryos of the zebra finch and society finch (Bengalese finch) suggested that they are younger than EGK-X (Yamasaki and Tonosaki, 1988; Agate et al., 2009; Murray et al., 2013). Due to the difficulty in retrieving pre-ovipositional (
We sought to identify serological markers capable of diagnosing preeclampsia (PE). We performed serum peptide analysis (liquid chromatography mass spectrometry) of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100%) and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%). The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA), 1 from alpha-1-antitrypsin (A1AT), 1 from apolipoprotein L1 (APO-L1), 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), 2 from kininogen-1 (KNG1), and 1 from thymosin beta-4 (TMSB4). We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension.
